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Abstract

Cellular resolution three−dimensional (3D) visualization of defined, fluorescently labeled long−range neuronal networks in the uncut adult mouse brain has been elusive. Here, a virus−based strategy is described that allowed fluorescent labeling of centrifugally projecting neuronal populations in the ventral forebrain and their directly, monosynaptically connected bulbar interneurons upon a single stereotaxic injection into select neuronal populations. Implementation of improved tissue clearing combined with light−sheet fluorescence microscopy permitted imaging of the resulting connectivity maps in a single whole−brain scan. Subsequent 3D reconstructions revealed the exact distribution of the diverse neuronal ensembles monosynaptically connected with distinct bulbar interneuron populations. Moreover, rehydratation of brains after light−sheet fluorescence imaging enabled the immunohistochemical identification of synaptically connected neurons. Thus, this study describes a method for identifying monosynaptic connectivity maps from distinct, virally labeled neuronal populations that helps in better understanding of information flow in neural systems